A tetravalent RGD ligand for integrin-mediated cell adhesion

Abstract

Monovalent RGD (arginine-glycine-aspartic acid) peptides or polymers furnished with RGD in random distributions are employed as cell-scaffolds and gene delivery vehicles. However, integrin binding to RGD is dependent on the spatial distribution (clustering) of the ligand and intrinsic integrin affinity via conformational changes (avidity). Here we have designed and expressed a polypeptide consisting of a tetrameric coiled coil and spacer facilitating polyvalent (clustered) display of integrin ligands; the RGD motif was used as proof of principle. Size-exclusion chromatography and circular dichroism showed that the polypeptide self assembled as a tetramer in solution with a defined secondary structure. Cell adhesion to surfaces coated with the polypeptide was up to 3-fold greater than that for (monovalent) RGDS peptide at equivalent concentrations. Moreover, the polypeptide in solution at concentrations ‡ 1 M inhibited cell adhesion to fibronectin-coated surfaces, while RGDS peptide in solution at concentrations up to 500 M did not. These cell data demonstrate that the polypeptide bound integrin receptors in a polyvalent manner. The polypeptide will therefore be of use in the engineering of tissue-culture scaffolds with increased cell adhesion activity, or to targeted gene delivery vehicles, and could incorporate protein ligands in place of the RGD motif.

Oligomerisation and thermal stability of polyvalent integrin α5β1 ligands

“…Since FIII9'-10-dimer appeared to form both dimers and trimers it was not possible to calculate the K d using this model. These values agree well with the dissociation behaviour inferred from cell adhesion data for a related tetrameric polypeptide assembly (equimolar concentrations at concentrations < 0.5 µM) [11]. However, precise K d values for coiled coils cannot always be easily assigned as we have also noted for the dimer [26].…”

“…Previously, very large chimeras have been studied employing four type III FN domains (FIII7-10), a large spacer of six titin repeats (58 kDa) and coiled coil [9]. In contrast, we have designed much smaller and more flexible, clustered α5β1 integrin ligands consisting of: the 9 th -10 th type III FN domain pair (FIII9'-10, a L1408P mutant with increased conformational stability and cell adhesion activity [10]), a spacer derived from the IgGhinge (previously shown to be sufficient to prevent steric clashes between bound integrins [11]) and five heptad repeats based on the GCN4 leucine zipper (with isoleucine and leucine variously placed in positions a and d to promote di-, tri-and tetramerisation [4]). For controlled immobilisation of these chimeras to a substrate or surface a unique C-terminal cysteine was introduced to facilitate either selective biotinylation via a thioether bond, followed by binding to avidin-coated surfaces, or direct covalent binding via the free thiol to gold surfaces [12].…”

18F-labeled mini-PEG spacered RGD dimer (18F-FPRGD2): synthesis and microPET imaging of αvβ3 integrin expression

“…18 F labeling of cyclic RGD peptide was first reported by Haubner et al, and the tracer 18 F-galacto-RGD exhibited integrin α v β 3 -specific tumor uptake in the integrin-positive M21 melanoma xenograft model [5]. In the clinical setting, 18 F-galacto-RGD also showed tumor uptake in certain cancer patients, yet the standardized uptake values were suboptimal owing to the relatively low α v β 3 binding affinity of the monomeric RGD peptide and the imperfect pharmacokinetics [6].…”

“…In the clinical setting, 18 F-galacto-RGD also showed tumor uptake in certain cancer patients, yet the standardized uptake values were suboptimal owing to the relatively low α v β 3 binding affinity of the monomeric RGD peptide and the imperfect pharmacokinetics [6]. Therefore, we and others have developed a series of dimeric and multimeric RGD peptides to improve the integrin α v β 3 targeting efficacy [7][8][9][10][11][12][13][14][15][16][17][18][19]. One tracer in particular, 18 F-fluorobenzoyl-E[c(RGDyK)] 2 ( 18 F-FB-E[c(RGDyK)] 2 , denoted as 18 F-FRGD2, Fig.…”

“…Therefore, we and others have developed a series of dimeric and multimeric RGD peptides to improve the integrin α v β 3 targeting efficacy [7][8][9][10][11][12][13][14][15][16][17][18][19]. One tracer in particular, 18 F-fluorobenzoyl-E[c(RGDyK)] 2 ( 18 F-FB-E[c(RGDyK)] 2 , denoted as 18 F-FRGD2, Fig. 1a), exhibited excellent integrin α v β 3 -specific tumor imaging with favorable in vivo pharmacokinetics [9,10].…”